EP0438497A1 - Vasopermeability-enhancing conjugates. - Google Patents
Vasopermeability-enhancing conjugates.Info
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- EP0438497A1 EP0438497A1 EP89911955A EP89911955A EP0438497A1 EP 0438497 A1 EP0438497 A1 EP 0438497A1 EP 89911955 A EP89911955 A EP 89911955A EP 89911955 A EP89911955 A EP 89911955A EP 0438497 A1 EP0438497 A1 EP 0438497A1
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- Prior art keywords
- conjugate
- agent
- delivery vehicle
- tissue
- tumor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
Definitions
- This invention relates to the use of immunological agents and other agents with unique specificities in vivo , and particularly, to means for enhancing the penetration and "binding of monoclonal antibodies and other macromolecules used for the diagnosis and therapy of human disease.
- mAbs tumor-specific monoclonal antibodies
- the effectiveness of antibodies to tumor-associated antigens in the therapy of cancer depends on the ability of antibodies to destroy their target cells by either direct cytotoxicity or complement-mediated cellular lysis. Complement-mediated lysis is triggered when the Clg component of the classical complement pathway binds to the Fc portion of antibodies bound to the surface of tumor cells, leading to the formation of the membrane attack complex. Tumor-bound antibodies can also recruit the natural defenses of the host by interacting with effector cells which themselves lyse the target. However, despite their multiple cytotoxic capacity, the actual experimental use of mAbs alone as cytotoxic agents has been unsatisfactory.
- conjugates of antibody and cobra venom factor are conjugates of antibody and cobra venom factor
- CVF is a glycoprotein, having the properties of the
- C3b, C3/C5 convertase of the alternative pathway of complement.
- CVF unlike its native analog, is not inactivated by complement control proteins. The presence of CVF on cell-bound antibody initiates assembly of the membrane attack complex and thereby cell death.
- immunoconjugates comprising monoclonal - - antibody and interferon, in which interferon enhances target cell lysis by activation of pre ⁇ existing cellular immune mechanisms, including natural killer (NK) cells.
- NK natural killer
- the circulatory system comprises the blood vascular system and the lymphatic system.
- the blood vascular system which distributes nutritive materials, oxygen, hormones and other substances to all parts of the body while removing the products of cellular metabolism, includes the heart and a series of tubular vessels: the arteries, veins, and capillaries.
- the arteries which by branching constantly increase in number and decrease in caliber, conduct blood from the heart to the capillary bed.
- the capillaries where the interchange of elements between the blood and the other tissues takes place, form a mesh- work of anastomosing tubules. Veins, in turn, return blood from the capillaries to the heart.
- the capillaries are typically comprised of simple endothelial cells that connect the arterial and venous sides of the circulatory system.
- Meshes of the capillary network are present throughout the body, varying in size and in shape in different tissues and organs. The intensity of metabolism in a region generally determines the closeness of the mesh. Therefore, there is a close network in the lungs, liver, kidneys, mucous membranes, glands, and skeletal muscle, as well as in the grey matter on the brain.
- the network has a large mesh and is sparse in tissues such as tendons, nerves, smooth muscle, and serous membranes.
- permeability The ability to transfer substances through the wall of capillaries is referred to as permeability. Permeability varies regionally and, under changed conditions, locally.
- tumor vessels are anatomically heterogeneous structures. Often, they consist of relatively undifferentiated channels, lined by a simple endothelium and with fewer pericytes and smooth muscle cells than would be expected of comparably sized vessels in normal tissues.
- the functional properties of tumor vessels have been more controversial; tumor vessels have been reported to be either more or less responsive to vasoactive mediators than normal vessels. (See, e.g., Hori, K. , et al., J. Natl. Cancer Inst. 74: 453-459 (1985).)
- One property of tumor vessels on which most investigators agree, however, is that, relative to normal vessels, tumor vessels are hyperpermeable to circulating macromolecules.
- Tumor cells grow radially from a central core of cells, rapidly outgrowing their blood supply, and leaving a necrotic, hypoxic core. In this instance, the distance from tumor cells to the nearest capillary is about 100 to 150 , a distance great enough to produce significant hypoxia and a perfusion deficit. These hypoxic cells show resistance to radiation and in addition, are inaccessible to injected drugs or antibodies. (Kaelin, W. et al.. Cancer Research 44: 896-899 (1984); Thomlinson, P. and Gray, L. , Br. J. Cancer 9: 539-549 (1955)).
- FIGURE 1 illustrates the effect of Lym/IL-2 immunoconjugate on radiolabeled Lym-1 F(ab')2 mice uptake in Raji-bearing nude mice.
- FIGURE 2 illustrates the effects of coinjection of IL- 2 and Lym-l/IL-2 vasoconjugate in lymphoma-bearing nude mice.
- FIGURE 3 illustrates the effects of Lym-l/IL-2 vasoconjugate predose escalation on tumor uptake by 1-125 Lym-1 F(ab')2 tracer.
- FIGURE 4 illustrates the effects of time of administration of Lym-l/IL-2 vasoconjugate in lymphoma- bearing nude mice.
- the invention provides immunoconjugates comprising biologically active agents, capable of provoking a vasoactive response, which are linked to monoclonal antibodies (mAbs) .
- the mAbs have the ability, when administered in vivo to a host, of preferentially binding to neoplastic tissue such as tumor cells or tumor cell ghosts.
- the biologically active agent is in this way localized at the site of neoplastic tissue where it provokes a response in which, by means of vasodilation and increased vasopermeability, or through the mechanism of an inflammatory response, the local circulation and blood supply to the tumor tissue are improved.
- the expansion of the blood volume within the tumor allows therapeutic and diagnostic agents, subsequently introduced into the host, to penetrate the tumor more completely and thus to be delivered in a larger and more effective dose.
- vasoactive conjugates preliminary to some types of immunotherapy will not only potentiate , deleterious side effects in the use of antibody conjugates comprising cytopathic substances such as anti-neoplastic drugs, toxins, or radionuclides.
- Such antibody conjugates remaining unbound in the circulation may lead to the unintended destruction of normal tissue, particularly tissues of organs of the renal, hepatic and reticuloendothelial systems which must eliminate them from the body.
- vasoactive conjugates make it possible to use a lower effective dose thereby reducing the amount of unbound circulating cytotoxic agent, and the risk to normal tissue.
- conjugates comprising vasoactive agents linked to monoclonal antibodies or, in a further extension, to other moieties (e.g., macromolecules or liposomes) that localize to permeable vessels. Therefore, while the term “immunoconjugates" may be used throughout the disclosure, it is to be expressly understood that a conjugate comprised of at least one immunoactive moiety is but one example of the diverse array of conjugates contemplated by the present invention.
- the monoclonal antibodies are selected for the ability, when administered intravenously ("I.v.”), of preferentially binding to tumors or to blood vessels and related structures in areas of inflammation, or where blood vessels are structurally abnormal at the tumor site.
- the vasoactive agent is in this way localized selectively at the sites of tumor or inflammation, where it provokes further increases in permeability. Such increases are selective for the sites and serve to facilitate passage of subsequently administered I.V. therapeutic agents from blood to tissue at the sites.
- Selective permeability enhancement induced by these vasoactive antibody conjugates, serves to increase the portion or dose of agents administered I.V. that reach the s e es re u c ac on. o on y potentiate therapy, but will substantially reduce the risk of deleterious side effects due to toxic metabolites or due to the development of im unological hypersensitivity responses.
- pharmaceutical conjugate comprising a delivery vehicle having the ability to localize at the site of neoplastic tissue, and an agent bound to the delivery vehicle acting to increase the blood supply to the neoplastic tissue.
- the conjugate is of sufficient size to be unable to penetrate normal, healthy vascular endothelium, but able to penetrate the vascular endothelium of tumor tissue.
- the agent acts to increase vasopermeability at an active site in vascular endothelium, or acts to provoke or exacerbate a local inflammatory reaction at an active site in vascular endothelium.
- a vasoconjugate in combination with an antineoplastic radioisotope or an antineoplastic toxin suggests a vasoconjugate in combination with an antineoplastic radioisotope or an antineoplastic toxin.
- the delivery vehicle comprises a macromolecule or particle having a molecular weight (MW) between 30,000 and 200,000.
- the present invention suggests a method for the diagnosis of neoplastic tissue, comprising administering to a host having the tissue an effective amount of a delivery vehicle having the ability to concentrate at the site of the tissue, the antibody being conjugated to an agent which acts to increase the blood supply to the neoplastic tissue, and contemporaneously or thereafter administering to the host a tumor imaging agent.
- the diagnostic agent is administered as a conjugate, comprising a delivery vehicle having the ability to concentrate at the site of the tissue, conjugated to the tumor imaging agent.
- immunoconjugates comprising monoclonal antibody av ng e a y o oca ze o umors or n e v c n y of structurally abnormal blood vessels, new vessels, or inflamed blood vessels at the tumor site; these conjugates further contain selected vasoactivators.
- the monoclonal antibody has specificity for subendothelial components of the blood vessel wall that become accessible to circulating antibody in inflamed vessels and in structurally abnormal vessels such as those found in tumors.
- target antigens include fibronectin, laminin, and type IV collagen.
- the antibodies have specificity for components of the coagulation cascade that are activated in the wall, or in the immediate perijacent environment of inflamed blood vessels, or in the necrotic areas of the tumor.
- antigens include fibrin, thrombin, and components of the complement system, and antibodies are available with these specificities.
- Yet another embodiment would employ antibody with specificity for antigens selectively expressed upon endothelial cells in inflamed blood vessels, but not in normal vessels.
- antigens would include various cell adhesion molecules that have been identified as responsible for adherence of polymorphonuclear leukocytes to inflamed blood vessel walls.
- the blood coagulation product fibrin is a particularly favored target for this approach. Fibronectin, which is distributed in a subendothelial distribution in blood vessels and is revealed by structural abnormality or by permeability # change, is another focused target for this approach.
- Another embodiment suggested by the present invention is the chemical linkage of a vasoactive moiety to a mAb with specificity to the tumor.
- the mAb would act to locate the vasoactive moiety to the tumor site during or after binding to tumor cells within the tumor.
- the vasoactive moiety would then act on the surrounding vessels in the immediate area of mAb binding.
- Another embodiment suggested by the present invention is the linkage of a delivery vehicle to a vasoactive agent a o ecu a — .e., a e co n o a "cassette" to be inserted into an organism, said cassette including, at a minimum, the genes coding for the delivery vehicle and the vasoactive peptide.
- the cassette could, in another embodiment, also include regulatory sequences.
- the cassette could be inserted into the genome of the organism, into a plasmid, or into a vector such as a virus or retrovirus, for example.
- This invention further discloses at least three antigen markers for tumor vasculature or "leaky” vasculature that emulates new tumor vasculature, and suggests means of utilizing same to construct delivery vehicles capable of specific localization to tumor sites, inflamed tissues, abscesses, and similar sites containing "leaky” vessels. Therefore, according to one aspect of the invention, there are provided immunoconjugates comprising a monoclonal antibody having the ability to localize at the site of neoplastic tissue (mAb) , and a vasoactive agent bound thereto.
- mAb neoplastic tissue
- the mAb has a specificity for tumor cells, and in a particularly preferred embodiment, the mAb has specificity for antigens associated with B-cell lymphoma cells.
- the monoclonal antibody may be Lym-1 or Lym-2.
- the vasoactive agent comprises a peptide, and in a preferred embodiment the peptide is a tachykinin. In a particularly preferred embodiment, the tachykinin is selected from the group consisting of phyllomedusin, physalaemin, and substance P.
- the vasoactive peptide comprises a leukotriene. In a particularly preferred embodiment, the leukotriene is selected from the group consisting of B4, C4, D4, and E4.
- the vasoactive peptide comprises an anaphylatoxin.
- the anaphylatoxin is selected from the group consisting of C3a and C5a.
- the lymphokine is selected from the group consisting of interleukin-1, interleukin-2 and tumor necrosis factor.
- the vasoactive peptide is the chemotactic factor ECF-A.
- the vasoactive peptide is an inflammagen.
- the inflammagen is selected from the group consisting of mastoparan and bestatin.
- the vasoactive peptide is a protease.
- the protease is selected from the group consisting of trypsin, chymase and thrombin.
- the vasoactive agent is a vasoactive carbohydrate.
- the carbohydrate is selected from the group consisting of glucan and proteoglucans.
- the vasoactive agent is a lipid.
- the lipid is selected from the group consisting of platelet-activating factor and prostaglandins.
- the lipid may be derivatized as the drug, Viprostol.
- the vasoactive agent is a biological amine.
- the amine is histamine.
- the mAb of the immunoconjugate may be an intact immunoglobulin.
- the mAb may be an immunoglobulin fragment consisting of the monovalent HL isoform.
- the mAb is one from which the Fc portion has been removed.
- the mAb is in the form of the F(ab , )2 portion.
- a method for treating a tumor comprising administering a vasoactive immunoconjugate to a tumor host, wherein the immunocon ugate comprises a mAb or other delivery vehicle having the ability to localize at the site of neoplastic tissue, allowing the immunoconjugate to bind to tumor tissue and allowing for the vasoactive effect of the immunoconjugate to occur, and either simultaneously or thereafter administering a therapeutic agent to the tumor host.
- the administered therapeutic agent is a cytotoxic chemical agent.
- the administered therapeutic agent is a cytotoxic immunological agent.
- a method for diagnosing a tumor comprising administering a vasoactive immunoconjugate to a tumor host, wherein the immunoconjugate comprises an mAb having the ability to concentrate at the site of neoplastic tissue, allowing the immunoconjugate to bind to tumor tissue and allowing for the vasoactive effect of the immunoconjugate to occur, and then either contemporaneously or thereafter administering to the host an immunodiagnostic agent.
- conjugates comprising a delivery vehicle , having the ability to localize at the site of neoplastic tissue, and an agent bound to the delivery vehicle, the agent acting to potentiate the action of a different antineoplastic agent against the tissue by increasing the blood supply thereto.
- Another embodiment suggests a conjugate of sufficient size to be unable to penetrate normal, healthy vascular endothelium, but able to penetrate the vascular endothelium of tumor tissue.
- the agent acts to increase vasopermeability at an active site in vascular endothelium, while yet another embodiment of the present invention suggests that the agent acts to provoke or exacerbate a local inflammatory reaction at an active site in vascular endothelium.
- the agent may comprise, for example, a drug, a vasoactive , , .
- the conjugate may comprise, for example, a carbohydrate, such as a glucan or proteoglucan, or it may comprise a lipid, such as platelet activating factor or 5 prostaglandins.
- Preferred 0 delivery vehicles include, without limitation, the F(ab')2, F(ab), or HL fragments of an immunoglobulin molecule, dextrans, monoclonal antibody, or liposomes.
- the liposomes have a diameter on the order of 80 nm, and the dextrans are high molecular weight 5 dextrans (70-150 KD) .
- the dextrans selectively localize in the walls of permeable vessels.
- the delivery vehicle has specificity for subendothelial components of the blood 0 vessel wall that become accessible to circulating antibody in inflamed vessels and in structurally abnormal vessels such as those found in tumors.
- Suggested components include, without limitation, fibronectin, laminin, and type
- a further embodiment discloses a delivery vehicle with specificity for components of the coagulation cascade that are activated in vascular walls, in the immediate perijacent environment of inflamed blood vessels, or in the necrotic areas of the tumor.
- the components comprise fibrin, thrombin, and components of the complement system.
- the delivery vehicle has specificity for antigens selectively expressed in or upon endothelial cells in inflamed vascular tissue such as that 35 found in the vicinity of tumors, but not in non-inflamed vascular tissue.
- Antigens suggested by the present invention include cell adhesion molecules responsible for adherence of polymorphonuclear leukocytes to inflamed vascular tissue, fibrin, fibronectin, fibrin degradation products, cell enzymes, platelets, and platelet products.
- the enzymes include peroxidases or other proteins that are released in necrotic or inflamed tissues.
- the present invention also suggests a method for the treatment or diagnosis of neoplastic tissue, comprising administering to the host of the tissue an effective amount of a delivery vehicle having the ability to concentrate at the site of the tissue, the antibody being conjugated to an agent which acts to potentiate the action of a different antineoplastic agent against the tissue by increasing the blood supply thereto, and contemporaneously or thereafter administering to the host a second conjugate, comprising a delivery vehicle having the ability to concentrate at the site of the tissue, conjugated to a therapeutic or diagnostic agent.
- Another embodiment suggests a method for immunotherapy of neoplastic tissue, comprising administering to a tumor host an effective amount of a conjugate referred to herein, and contemporaneously or thereafter administering to the tumor host a delivery vehicle having the ability to concentrate at the site of the tissue and directed to the therapy thereof.
- a further embodiment discloses the conjugation of a tumoricidal agent to the delivery vehicle.
- Still another embodiment suggests a method for the immunotherapy of neoplastic tissue, comprising administering to a host of the tissue an effective amount of a conjugate referred to herein, and contemporaneously or thereafter administering to the host a pharmacological agent directed to the therapy of the tissue.
- a method for the immunodiagnosis of neoplastic tissue comprising administering to a host of the tissue an effective amount of a conjugate referred to herein, and contemporaneously or thereafter administering to the host a ability to concentrate at the site of the tissue and a detectable agent conjugated thereto.
- Another embodiment discloses a method for immunotherapy of inflamed tissue, comprising administering to a tumor host an effective amount of a conjugate referred to herein, and contemporaneously or thereafter administering to the tumor host a second delivery vehicle having the ability to concentrate at the site of the tissue and directed to the therapy thereof.
- a method for the immunotherapy of inflamed tissue comprising administering to a host of the tissue an effective amount of a conjugate referred to herein, and contemporaneously or thereafter administering to the host a pharmacological agent directed to the therapy of the tissue.
- Another aspect of the present invention suggests a method for constructing a conjugate for pharmaceutical use, comprising attaching a delivery vehicle having the ability to localize at the site of neoplastic tissue or nucleotides coding for same, to at least one agent acting to increase the blood supply to the neoplastic tissue or nucleotides coding for same.
- the present invention further suggests a therapeutic kit, comprising a conjugate, comprising a delivery vehicle having the ability to localize at the site of neoplastic tissue, and an agent bound to the delivery vehicle acting to increase the blood supply to the neoplastic tissue, and an antineoplastic therapeutic agent.
- the present invention discloses a diagnostic kit, comprising a conjugate, comprising a delivery vehicle having the ability to localize at the site of neoplastic tissue, and an agent bound to the delivery vehicle acting to increase the blood supply to the neoplastic tissue, and a tumor imaging agent.
- a method for genetically constructing a conjugate comprising attaching at least one agent or nucleotides co ng or same o a eas one e very ve c e or nucleotides coding for same.
- vasoactive agents have been shown to induce more extensive changes in tumor vessels 0 than in normal vessels. (See, e.g.. Cater, et al., Br. Cancer 20: 517 (1966).) This effect can be maximized by linking vasoactive agents to monoclonal antibodies or other moieties that bind with molecules in the vascular wall, or in the immediate surrounding environment, of abnormal b ⁇ ood 5 vessels within tumors.
- This application is thus an extension of our previous application, as cited above, in which antibody with specificity for tumor cells was conjugated with vasoactive agents, with the goal of inducing permeability changes.
- the present application is thus an extension of our previous application, as cited above, in which antibody with specificity for tumor cells was conjugated with vasoactive agents, with the goal of inducing permeability changes.
- the antibodies to be used have the following properties. First, following chemical
- a potential therapeutic agent such as a drug or a monoclonal antibody
- injected intravenously shows preferential passage from the blood into tissue fluid at the abnormally permeable site.
- the percentage of a given dose of agent delivered to the tumor site has been multiplied from two to six fold in studies to be described within.
- This method may be utilized for improving delivery of anticancer agents to tumor sites, either drugs, monoclonal antibodies, or conjugates of monoclonal antibodies with drugs, toxin or radioisotopes.
- a further embodiment employs monoclonal antibodies to macromolecules that are exposed to the bloodstream in structurally abnormal vessels within necrotic areas in tumors or inflamed tissues.
- antigens include fibrin degradation products, and various cell enzymes such as peroxidases that are released by granulocytes or other cells in necrotic or inflamed tissues.
- vasoactive compounds for attachment to antibody are analogous to those described below, and include peptides, carbohydrates, lipids, and their derivatives.
- Another embodiment would employ antibody with specificity for antigens selectively expressed upon endothelial cells in inflamed blood vessels, but not in normal vessels.
- antigens would include various cell adhesion molecules that have been identified as responsible for adherence of polymorphonuclear leukocytes to inflamed blood vessel walls.
- the blood coagulation product fibrin is a particularly favored target for this approach. Fibrin is not normally present within the bloodstream, existing only as a circulating precursor molecule, fibrinogen, which has a molecular weight (MW) of approximately 340 kilodaltons (KD) . Likewise, fibrin is not present in normal tissue or tissue fluids.
- Fibrinogen is also absent from tissue fluids, since its high molecular weight precludes escape from the blood across normal, intact endothelium. permeability, fibrinogen may, however, escape into the tissues where it is rapidly converted to fibrin through activation of intravascular clotting mechanisms. Fibrin deposits thus form at the site of permeability change. In tumors, microdeposits of fibrin are particularly present in capillary sprouts and in the vicinity of blood channels that lack complete endothelial lining.
- fibrinogen serves as a marker of vascular leakage by virtue .of its molecular weight characteristics. Secondly, its detection is facilitated by its conversion into an insoluble product immediately upon escape from the vessel. Monoclonal antibodies directed against fibrin (that are non-reactive with fibrinogen) will therefore show selective homing to permeable vessels that have been "marked” by fibrinogen leakage and fibrin deposition.
- Fibronectin which is distributed in a subendothelial distribution in blood vessels and is revealed by structural abnormality or by permeability change, is another focused target for this approach. See, e.g., Christensen, et al.. Cancer, 1988; Dvorak, et al., NEJM 315: 1650 (1986); and Jain, Cancer Res. 48: 2641 (1988).)
- vasoactive conjugates may also prove efficacious, including those which improve the extravascular penetration and binding of monoclonal antibodies, as well as other drugs or molecules.
- conjugates disclosed herein have proven effective when large molecules are utilized, smaller molecules, such as chemotherapeutic drugs, may also exhibit increased penetration and binding.
- Embodiments using vehicles other than monoclonal antibodies employ macromolecules (molecular weight range: 70,000-1,000,000 or more) or microparticles, including liposomes, with a diameter on the order of 80 nanometers (nm) that localize to permeable vessels on the basis of their physio-chemical characteristics.
- dextrans MW 150 KD
- vasoactive agents vessels that show marginal permeability changes, thereby markedly enhancing permeability at the sites only.
- therapeutic modalities administered subsequently show a higher proportion of administered dose at the initial sites.
- the immunoconjugates of the invention are prepared by genetic approaches, or covalently or otherwise linking a selected clinically useful mAb to a selected biologically active agent which is inflammation provoking, and preferably vasoactive.
- the linking agent and the chemical procedure of assembling the immunoconjugate should be selected and carried out so as not to compromise the effectiveness of the antibody in binding to target cells or the effectiveness of the vasoactive agent in stimulating natural defense mechanisms.
- Suitable monoclonal antibodies for use in the invention comprise not only those having a specificity for antigens unique to the tumor cells, but also those having a shared specificity for antigens of normal tissues.
- the essential property is that these monoclonal antibodies be effective, according to the purpose of the invention, as carriers which preferentially concentrate vasoactive agents at the site of the tumor.
- Suitable monoclonal antibodies may be those having a specificity to antigens, such as intercellular substances, that are either more abundant or more easily bound in tumor tissue than in normal tissue.
- One example is antibody to nuclear antigens, as disclosed in U.S. Patent No. 4,861,581.
- mAbs against tumor or normal cellular antigens suitable for use in the immunoconjugates of the invention, are available commercially (Centocor, Malvern, PA; Hybritech, San Diego, CA) . Others may be prepared according to the well- established hybridoma procedure of Kohler and Milstein, (Nature 256: 495 (1975)), and hybridoma cell lines, splenocytes from mice immunized with tumor antigen are fused with cells from a non-secreting mouse myeloma fusion line, such as P3X63-Ag8.653 (American Type Culture Collector, Rockwell, MD) , according to kit instructions, for example, HyBRL Prep Kit (Bethesda
- the fused hybridomas cells are then transferred into the wells of microtiter plates where they are grown for several days.
- the supernatants in the wells are tested for production of mAbs to tumor or cellular antigens by any convenient immunoassay, for example, an ELISA, and the positive hybridoma cell lines, that is, those producing acceptable mAbs, are expanded into permanent culture.
- MAbs may be purified from the supernatants of these cultures by gel chromatography, for example, using the Affi-Gel Protein A column (Bio-Rad,
- mAbs specific for lymphoma cells Lym-1 and Lym-2, are used (Techniclone International, Inc., Tustin, California) .
- the suitability of tumor-specific mAbs for in vivo use is determined by the biodistribution, cellular localization, selective binding, and rate of clearance from the tumor host, or an animal model of the tumor host.
- the performance of the assembled immunoconjugates may also be determined by parallel studies. Studies to assess this suitability are conveniently carried out by means of labeled mAbs, for example, l3 ll-mAbs, radioiodinated, for example, by the modified Chloramine-T procedure of McFarlane, A., Biochem. J. 62:135-143 (1956).
- the immunoreactivity of radiolabeled anti-tumor mAbs may be determined by an in vitro live cell radioimmunoassay procedure as described in Example 1 for the Lym-1 and Lym-2 mAbs (see Epstein, A. et al.. Malignant Lymphomas and Hodcfkin / s Disease: Experimental and Therapeutic Advances. Martinus Nijoff Publ. Co., Boston (1985), pp. 569-577).
- the effectiveness of an anti-tumor mAb in vivo may be evaluated by appropriate radioimaging, biodistribution, histological studies, and autoradiographic methods performed after injecting the tumor-bearing host with the labeled mAb.
- the ability of the mAb to concentrate selectively at the tumor site is determined by radioimaging.
- Posterior gamma scintillation images (100,000 cpm) are obtained from the anesthetized host on alternate days after injection of the radiolabeled mAb, using a gamma scintillation camera with a pinhole collimator.
- the camera is preferably interfaced to a computer system.
- An appropriate 13 l j standard with the • same activity is counted to quantitate the data.
- the host animal is sacrificed and blood, major organs and tumor tissue excised, weighed, and counted to determine the biodistribution of the mAb.
- tumor tissue may be fixed and embedded, and tissue sections examined by autoradiography to determine the radiolabeled mAb bound to the tumor.
- the mAb of the immunoconjugate may be either intact whole antibody, the monovalent HL isoform, the F(ab')2 portion of antibody, or Fab antibody fragments. Removal of all or part of the Fc portion of the antibody molecule can facilitate its use by removing sites or domains which interact with non-tumor components such as Fc receptors or complement while leaving the antigen binding sites intact.
- Antibody fragments such as Fab, HL, and F(ab')2, which have 1/3, 1/2 and 2/3 the weight of whole antibody respectively, have the ability to cross capillary walls and diffuse through the interstitial tissue more readily, and so are able to diffuse more rapidly into the tumor.
- Fab fragments may be prepared by digestion of whole antibody with papain, or digestion of whole antibody to F(ab')2 fragments with pepsin, followed by digestion of interchain disulfide bonds to yield univalent fragments. (See Porter, R. , Biochem. J. 73; 119 (1959).) HL fragments may be derived according to the technique set forth in Nature 194: 355 (1962) or PNAS fUSA ⁇ 50: 314-321 (1963). 2. Macromolecules or Microparticles
- vasoactive immunoconjugates of the present invention are distinct from drug or toxin immunoconjugates in their mode of action. Drug and toxin conjugates are used to kill tumor cells directly. Vasoactive conjugates are used to increase the flow of blood and/or the vessel permeability in the tumor so as to improve the extravascular penetration and binding of monoclonal antibodies and other drugs or molecules in vivo .
- Inflammation can be induced by chemotactic factors which attract polymorphonuclear leukocytes, macrophages, eosinophils, basophils, mast cells, T-cells and other cells associated with inflammation. These cells, when stimulated, secrete immunomodulatory factors which then act on the tumor blood flow and blood vessel permeability to increase the percent of the injected dose penetrating and binding to the tumor.
- Vasoactive agents having the described reactivity at the tumor site and suitable for linking to monoclonal antibodies in an immunoconjugate are found in several biochemical classes, including peptides, carbohydrates, and lipids, and their derivatives.
- Tachykinins are a family of deca-, enceda-, and dodeca- peptide amides, having a phenylalanine (Phe) residue at position 5 from the COOH terminus. They have potent pharmacological effects on blood pressure, non-vascular smooth muscles, and the exocrine glands (Erspamer, V., TINS. Nov. 1981, pp. 267-269) .
- Substance-P a mammalian tachykinin, promotes vasodilation and plasma extravasation through antidromic stimulation of chemosensitive nerve fibers (Lambeck, F. and Halzer, P., Naunyn-Schmeideberg's Arch. Pharmacol. 310: 175-183 (1979)). Substance-P also mediates histamine release from tissue mast cells (Hagermark, O. et al., J. Invert. Dermatol. 71: 233-235 (1978)). In preferred embodiments of the invention, Substance-P and an amphibian analog, physalaemin, are conjugated to clinically useful Mabs for use in promoting the dilation of the tumor microvasculature.
- leukotrienes are sulfidopeptides which are potent mediators in atopic allergy.
- the action of these mediators on blood vessels with its associated inflammatory action is responsible for the clinical manifestations and physical features of the disease.
- leukotrienes B4, C4, D4 and E4 are conjugated to clinically useful mAbs for use in producing a local inflammatory reaction at the tumor site.
- Anaphylatoxins are peptide fragments released during activation of serum complement. Enzymatic cleavage of pro C3a and C5a, respectively. These peptides have been designated anaphylatoxins because of their ability to produce a reaction that resemble anaphylactic shock. Both C3a and C5a have the ability to increase vascular permeability and to release granules containing serotonin and histamine from tissue mast cells. C5a, in addition and perhaps cooperatively with C3a, is chemotactic, inducing the migration and aggregation of neutrophils. (See Nagata, S. et al.. Int. Arch. Allergy Appl. Immun.
- C3a, C5a, or their biologically active peptide sequences are conjugated to tumor-specific Mabs and used to produce a localized inflammatory response at the tumor site as an alternative approach to enhance the extravascular penetration of monoclonal antibodies.
- Lymphokines comprising the interleukins IL-1 and IL-2 and tumor necrosis factor (TNF) , are endogenous stimulators of the immune response that act and interact in complex roles in the defense of an organism against foreign agents.
- TNF tumor necrosis factor
- IL-2 is of particular interest for use in im unoconjugates.
- This lymphokine has no anti-tumor activity of its own, but appears to have potent activity when administered with lymphokine-activated killer (LAK) cells.
- LAK lymphokine-activated killer
- Its use as an anti-tumor agent appears to be limited, because its ability to mediate vascular permeability and -extravasation in the host produces severe side effects due to retention of fluid.
- LAK lymphokine-activated killer
- the vasoactive properties of IL-2 are well suited to its use in the immunoconjugates of this invention.
- small synthetic oligopeptides comprising the functional region of interleukin, can also be suitable for use in the immunoconjugates.
- immunoconjugates See, e.g., Antoni, G. et al., J.
- peptides suitable for use in the vasoactive immunoconjugates are the human eosinophil acidic tetrapeptides (ECF-A) , Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu, which have the ability through chemotaxis to promote a local eosinophilia (Turnball, L. et al.. Immunology 32: 57- 63 (1977)).
- ECF-A human eosinophil acidic tetrapeptides
- Val-Gly-Ser-Glu Val-Gly-Ser-Glu
- Ala-Gly-Ser-Glu the ability through chemotaxis to promote a local eosinophilia
- certain peptides, the inflammagens when used in vasoactive immunoconjugates, would be capable of degranulating mast cells at the tumor site, releasing histamine and provoking a local inflammatory response.
- mastoparan is a tetradecapeptide isolated from wasp venom (Okano, Y. et al.. Fed. Europ. Biochem. Soc. 188(2): 363-366 (1985)).
- mastoparan either isolated from the natural source or produced synthetically is linked to a tumor- specific mAb. (See Hirai, Y.. et al., Chem. Pharm. Bull. 27(8) : 1942-1944 (1979).)
- Proteases released from mast cells upon immunologic activation appear to provoke hypersensitivity reactions in skin.
- the possible actions of these proteases include digestion of the blood vessel basement membrane with resultant increased vascular permeability and the influx of secondary inflammatory cells.
- Tryptase an endopeptidase similar to pancreatic trypsin, is a tetramer composed of two 35 k lo a on an two 37 o a on su un s. s the principal protease of human lung mast cells and is present in mast cells from all locations.
- Chymase found in human skin mast cells, has a specificity like that of pancreatic chymotrypsin. (Serafin, W. and Austin, K. , NEJM.
- tryptase and chymase are conjugated to tumor-specific mAbs for use in producing a local inflammatory reaction at the tumor site.
- Certain lipid compounds can be effective as immunoconjugates.
- platelet-activating factor is the vasoactive agent of the immunoconjugate.
- PAF is a phospholipid produced by human neutrophils which appears to be a potent mediator of the immune response.
- PAF is linked with virtually all inflammatory and immune processes, for example, with respect to the vasoactive peptides listed above, PAF stimulates Substance-P release, and induces the formation of other vasoactive agents, such as leukotrienes or prostaglandins. Its use in immunoconjugates can amplify the effect of these other agents whether endogenous or used in complementary immunoconjugates.
- the hypotensive agent, Viprostol, a prostaglandin derivative (American Cyanamid, Pearl River, NY) is the active agent in the immunoconjugate. Viprostol lowers arterial blood pressure mainly through vasodilation. (See Chan, P. et al., J. Hypertension 4(6): 741-746 (1986).)
- Use of a tumor-specific, targeted Viprostol dose will dilate the vasculature of the tumor to expand blood volume therein.
- the natural prostaglandins (PGE's) , or synthetic analogues which are known to possess hypotensive effects, can be effectively used.
- PGE's prostaglandins
- Histamine a component of mast cell granules released upon immune stimulation, acts through two types of receptors, designated Hi, and H2, to produce, among other effects, increased venular permeability and vasodilation as described for the leukotrienes.
- histamine is conjugated to tumor-specific mAbs for use in producing a local inflammatory reaction at the tumor site.
- the effective agents of the immunoconjugates are vasoactive carbohydrate compounds.
- the vasoactive carbohydrate is glucan.
- Glucan is a / 9-l,6 linked polyglucose derived from Saccharomyces cerevisiae which has a number of immunopotentiating effects (Glovsky, M. et al., J. Reticuloendothelial Society 33: 401-413 (1983)), but, unlike the interleukin IL-2, is non-toxic.
- Glucan appears to exert its effects by stimulating the complement system, generating, among other complement fragments, the vasoactive C3a and C5a peptides.
- Glucan targeted to tumors by means of specific mAbs, could act locally through C3a and C5a to dilate the tumor vasculature.
- Conjugant molecules are selected according to availability and applicability to the stated goals of therapy or study. Chemical Conjugation Methods
- the structural link between the mAb, macromolecule, or microparticle and the vasoactive agent, and the chemical method by which they are joined, should be chosen so that the binding ability of the mAb and the biological activity of the agent, when joined in the conjugate, are minimally compromised.
- the methods from which the most effective conjugation chemistry may be selected are the following: a) Carbodiimides may be regarded as anhydrides of . - - (ECDI) produces crosslinks between the antibody and the conjugant, regardless of either molecule's orientation.
- Conjugants are derived by condensation of the antibody and conjugant under acidic conditions with ECDI. This method provides a rapid and simple means of conjugation. (See Goodfriend, T. et al..
- the exposed terminal groups can then link to NH2 groups on proteins in a: S-chiff base linkage which is reduced with NaBH4.
- S-chiff base linkage which is reduced with NaBH4.
- NaBH4 Na-hydroxysuccinimide
- the use of periodate oxidation to conjugate glucan to an mAb is illustrated in Example 5.
- N-hydroxysuccinimide (NHS) activates a terminal COOH group, for -example, of a peptide, to form an active ester derivative that can be covalently coupled to the protein of the monoclonal antibody. This method has been used to attach 30 molecules of chlorambucil/antibody with little loss of binding activity. (Smyth, M. et al., J.
- the genetic sequence of the vasoactive peptides can be engineered into the sequence of the mAb as illustrated in Example 11.
- the immunoconjugates are evaluated in vitro by the proliferation radioimmunoassay described by Bindon et al. , Br. J. Cancer 42: 123-133 (1983), and illustrated in Example 8, to determine the degree of immunoreactivity and biological activity retained by the product. Only immunoconjugates found to have greater than 80% immunoreactivity as compared to the unconjugated antibody are used for in vivo experiments.
- a successful immunoconjugate will maximize the clinical effectiveness of monoclonal antibody-based diagnosis and therapy.
- the vasoactive immunoconjugate is given before or with the immunodiagnostic, chemotherapeutic, or immunotherapeutic dose so that the tumor vasculature will be made more susceptible to penetration by the effective agents therein.
- the time required to produce the maximum vasoactive effect depends on the specific conjugate chosen and its mechanism of action. It is anticipated that if given before the mAb doses the minimum time between administration of the vasoactive immunoconjugate and the administration of the diagnostic or therapeutic agent is at least about 20 minutes, and the maximum time is about 72 hours.
- the optimal interval between the time of administering the vasoactive immunoconjugate and the dose can be experimentally determined by animal studies or appropriate studies of the tumor host using labeled immunoconjugate with the imaging, biodistribution studies, and the histological methods described above.
- vasoactive immunoconjugate to be given is based on criteria of medical judgment and experience, both objective and subjective. However, an adequate measure of an effective dose is that amount required to increase localization of a subsequently administered diagnostic or therapeutic agent to an extent which improves the clinical efficacy of therapy, or accuracy of diagnosis, to a statistically significant degree. Comparison is made between treated and untreated tumor host animals to whom equivalent doses of the diagnostic or therapeutic agent are administered. Where applicable, for example in the use of diagnostic or therapeutic agents that are toxic to normal tissue, an effective dose of vasoactive conjugate is also that which similarly reduces such toxic effects.
- the immunodiagnostic dose may comprise mAb having a specificity for a tumor and having a label which is detectable in vivo .
- this label comprises a radioactive isotope.
- the immunotherapeutic dose may similarly comprise a clinically useful mAb.
- This mAb may further be attached to a tumoricidal agent, for example, a radioisotope, a chemotherapeutic drug or a toxin.
- Raji cells are washed twice in cold PBS containing 1 mg/ml bovine serum albumin and 0.02% sodium azide. (See, e.g., J. Nat- * ! Cancer Inst. 37: 547-559 (1966) for a description of Raji cells and methods of obtaining same.) Cells (5 x 10 5 ) resuspended in 100 ⁇ l of wash buffer are pipetted into microtiter wells (Immulon Removawell Strips; Dynatech Labs., Inc., Alexandria, VA) .
- microtiter plates are pretreated the previous night with BSA 10 mg/ml) in PBS with azide in order to prevent the antibody solutions from binding to the wells.
- BSA 10 mg/ml Commercially available mAbs specific for lymphoma cells, Lym-1 and Lym- 2, are available from Techniclone International, Inc.,
- Radiolabeled Lym-1 and Lym-2 are then added (100,000 cpm/well) in a volume of 100 ⁇ l/well and the plates are incubated for 30 minutes at room temperature with constant shaking. The plates are then washed 4 times by spinning at 1,000 rpm for 5 minutes, and aspirating the supernatants with a 12-tip micromatic manifold, and then resuspending the cells in 200 ⁇ l of wash buffer using a
- Titertek Multichannel pipet (Flow Labs, McLean, VA) .
- the wells are then separated mechanically and counted in a gamma counter to quantitate the amount of label binding to the cells.
- Physalaemin (Sigma Chemical Co., St. Louis, MO) is conjugated to monoclonal antibodies Lym-1 and Lym-2 by the carbodiimide method.
- Physalaemin, Lym-1 or Lym-2, and 1- cy ⁇ lohexyl-3-(2-morpholinoethyl 1) carbodiimide metho-p- toluene sulfonate (CDI) Aldrich Chemical Co., Milwaukee,
- WI are mixed in a 1:3.6:36 ratio by weight and incubated for 20 min .at pH 5.0 at room temperature. The reaction is terminated by dialysis against PBS, pH 7.2 overnight. The conjugate is purified by FPLC Superose (Pharmacia, Piscataway, NJ) column chromatography and stored at 4 ⁇ C in
- Monoclonal antibodies are cleaved to F(ab')2 fragments using pepsin so as to avoid non-specific binding to leukocytes by the Fc portion of the ant body.
- C3a (57-77) , containing an N-terminal cysteine residue, is synthesized using automated protein synthesis.
- the C3a peptide (1 mg) is dissolved in 300 ⁇ l of 4M guanadinium-PBS pH 7.5.
- the pH is adjusted by dialysis to between 3 and 4 with approximately 3 L of 17% H3PO4. This solution is placed in a receiving tube (17 X 100 mm) .
- the F(ab')2 antibody (60 nmole) is dissolved in 1.0 ml
- the conjugated antibody is stored at 4 ⁇ C.
- C5a is coupled to F(ab')2 antibody using the bifunctional ⁇ cross-linking reagents dimethyl . superimidate or SPDP. Conditions will be adjusted to produce 1/1 C5a-
- Viprostol can be derivatized by adding a cis-aconityl spacer arm through its 11-hydroxy group. In this reaction,
- Viprostol (5 mg) is dissolved in 1 ml of 0.1M Na2HP0 4 in a test tube and cooled in an ice bath. A molar excess (5 mg) of cis-aconityl anhydride (Aldrich chemicals, Milwaukee, WI) is added slowly to the solution while stirring, and the pH is kept between 8 and 9 by careful addition of IN NaOH. Thin-layer chromatography of samples is used to monitor the progress of the reaction. A tracer of 3 H- or l 4 C-labeled Viprostol may be added to the reaction mixture and the progress of the reaction monitored by autoradiography of the thin-layer plates. Separation and purification of the derivative may be achieved by using acidification and purification of the product or a column chromatography method.
- Viprostol to monoclonal antibodies, a solution of the cis-aconityl Viprostol derivative is added to a solution containing Lym-1 or Lym-2 monoclonal antibodies. The mixture is then allowed to incubate for 30 minutes at room temperature at pH 5.0. The reaction is terminated and the conjugate purified by eluting the sample through a Sephadex G-25 column, or by FPLC Superose column chromatography.
- Glucan is carefully oxidized by using periodate oxidation to cleave one of its sugar moieties without affecting its bioactivity, and using only a 1 to 2 molar excess of NaI ⁇ 4.
- the aldehyde groups produced in the glucan by oxidation will react with -NH2 groups on the monoclonal antibodies to form a Schiff base.
- the Schiff base linkage is then reduced with NaBH4, at a concentration of 0.3 mg/ml to form a stable amine linkage of glucan-Mab conjugate.
- An active ester of mastoparan is prepared by reaction with N-hydroxysuccinimide (NHS) in dimethylformamide and using N,N-dicyclohexylcarbodiimide (DCC) as a condensation reagent.
- a solution of the mastoparan active ester in DMF is then added to a solution containing Lym-1 or Lym-2 monoclonal antibodies at pH 7.0 and allowed to react for 1 to 2 hours at room temperature. Any undissolved reagents, mainly dicyclohexyl urea and/or precipitated protein, are removed by centrifugation.
- Free mastoparan and other unreacted starting materials can be removed by gel filtration chromatography using a Sephadex G-25 (Pharmacia, Piscataway, NJ) column.
- the amount of mastoparan incorporated in the conjugate is determined by means of a tracer of tritium (H 3 )-labelled mastoparan.
- Recombinant Interleukin-2 (rIL-2) (Cetus Corporation, Emeryville, California) is provided in vials containing 0.3 mg or 1.2 mg/vial.
- Purified monoclonal antibody such as Lym-1 is conjugated to rIL-2 using l-cyclohexyl-3-(2- morpholinoethyl carbodiimide metho-p-toluenesulfonate) ("CDI”) and N-hydroxysulfosuccinimide in a 1:2:50:50 ratio by weight to give a total volume of 0.3 ml in phosphate buffer, pH 7.4.
- CDI l-cyclohexyl-3-(2- morpholinoethyl carbodiimide metho-p-toluenesulfonate)
- CDI l-cyclohexyl-3-(2- morpholinoethyl carbodiimide metho-p-toluenesulfonate
- the soluble coupled antibody is chromatographed on a Sephadex G-100 column calibrated with blue dextran. Using this procedure, approximately 1-2 molecules of rIL-2 are coupled to each monoclonal antibody ("mAb") molecule.
- the immunoconjugate preparation is then adjusted to 1 mg/ml, sterile filtered, and stored at 4°C until use. This procedure can be used to couple rIL-2 to any tumor-specific monoclonal antibody.
- rIL-2 conjugate treatment may then be supplemented or f oll owed by use of conventional chemotherapeutic agents including, for example, cis- platinum, for the treatment of different types of cancers .
- the genetic sequence of the vasoactive peptide can be engineered into the sequence of the monoclonal antibody.
- mRNA coding for the anti- fibronectin monoclonal antibody is isolated. From this mRNA, a cDNA is synthesized for both the heavy and light chains of immunoglobulin. This cDNA is subsequently 1) amplified using the polymerase chain reaction; 2) sequen ⁇ ed; and 3) mapped by restriction endonu ⁇ leases. The appropriate DNA sequence of the vasoactive peptide, such as IL-2, is then ligated to the ends of the heavy chain gene in the constant region.
- the completed engineered gene is then reintroduced into a eukaryotic or prokaryotic expression system by gene transfeetion methods (for example, using electroporation or the calcium phosphate method) , so that the protein product is expressed in large scale cell culture.
- gene transfeetion methods for example, using electroporation or the calcium phosphate method
- two active IL-2 moieties will be part of each immunoglobulin molecule.
- the best site of attachment for each vasoactive peptide may be different and may easily be determined via experimental methods.
- the sequence for the vasoactive peptide can be ligated to either human or mouse immunoglobulin heavy chain sequences to produce human, mouse, chimeric or other species or combinations of immunoglobulin molecules.
- Fresh murine splenocytes are placed in culture with rIL-2 for 7 days after stimulation by PHA for 3 days. The cells are then washed free of rIL-2 and resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics. One hundred ⁇ l containing 10 5 cells are placed in triplicate in microtiter plate wells in the presence of 100 ⁇ l of varying concentrations of immunoconjugate (test sample) , rIL-2 (positive control) and Lym-1 or Lym-2 (negative control) .
- the x-axis dilution coordinate of the control sample which crosses this curve at the 50% maximum 1 25 I-IUDR uptake is defined at that value which corresponds to 1 unit of rIL-2 activity.
- y-axis coordinate The x-axis dilution coordinate of the control sample which crosses this curve at the 50% maximum 1 25 I-IUDR uptake
- Vasoactive Compounds to Increase Extravascular Tumor Penetration by Monoclonal Antibody Lym-1
- mice To test the relative effects of the IL-2 vasoactive immunoconjugate on the biodistribution and tumor uptake of Lym-1 in lymphoma-bearing nude mice, groups of five mice each bearing 0.5 g Raji lymphoma subcutaneous transplants were given intravenous doses of Lym-1 (control) or Lym- l/IL-2 immunoconjugate (experimental) at times zero or 2 1/2 hours before the administration of 20 ⁇ g of Lym-1 F(ab') 2 radiolabeled with 50 ⁇ Ci of 1-125. Three days later, all the mice were sacrificed and the tumors and normal ' organs removed to quantitate the amount of label per gram of tissue.
- mice receiving the experimental Lym-l/IL-2 vasoconjugate showed a 200% increase in mAb localization over appropriate controls (Fig. 1) .
- this increase in mAb localization enhances the tumor/blood ratio approximately twofold (Fig. 2) , is dose-dependent (maximum effect between 30-50 ⁇ g of vasoconjugate; Fig. 3) , and is time dependent (Fig. 4) with a maximum effect demonstrated 2 1/2 hours before the administration of the mAb.
- vasoactive immunoconjugates be used to enhance the delivery of 1) drugs or drug-containing liposomes, and 2) therapeutic monoclonal antibodies.
- the mechanism of action of the immunoconjugate is the production of an increase in the permeability and/or blood flow at the tumor site.
- the immunoconjugate is generally administered 1-3 hours before the therapeutic dose of drug, monoclonal antibody, or liposome is administered.
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WO1986003938A1 (en) * | 1985-01-11 | 1986-07-17 | The Regents Of The University Of California | Method for preserving liposomes |
US4771128A (en) * | 1986-10-10 | 1988-09-13 | Cetus Corporation | Method of purifying toxin conjugates using hydrophobic interaction chromatography |
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US4753894A (en) * | 1984-02-08 | 1988-06-28 | Cetus Corporation | Monoclonal anti-human breast cancer antibodies |
US4724213A (en) * | 1985-05-24 | 1988-02-09 | Northwestern University | Murine hybridoma Lym-1 and diagnostic antibody produced thereby |
US4724212A (en) * | 1985-05-24 | 1988-02-09 | Northwestern University | Murine hybridoma Lym-2 and diagnostic antibody produced thereby |
US4753895A (en) * | 1987-02-24 | 1988-06-28 | Hughes Aircraft Company | Method of forming low leakage CMOS device on insulating substrate |
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1989
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- 1989-10-11 JP JP1511059A patent/JP2961173B2/en not_active Expired - Lifetime
- 1989-10-11 WO PCT/US1989/004513 patent/WO1990003801A1/en active IP Right Grant
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1986003938A1 (en) * | 1985-01-11 | 1986-07-17 | The Regents Of The University Of California | Method for preserving liposomes |
US4771128A (en) * | 1986-10-10 | 1988-09-13 | Cetus Corporation | Method of purifying toxin conjugates using hydrophobic interaction chromatography |
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Also Published As
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DE68925974T2 (en) | 1996-11-14 |
AU627785B2 (en) | 1992-09-03 |
WO1990003801A1 (en) | 1990-04-19 |
AU4424589A (en) | 1990-05-01 |
JP2961173B2 (en) | 1999-10-12 |
EP0438497A4 (en) | 1992-02-26 |
JPH04503945A (en) | 1992-07-16 |
EP0438497B1 (en) | 1996-03-13 |
ATE135236T1 (en) | 1996-03-15 |
IL91933A (en) | 1994-12-29 |
DE68925974D1 (en) | 1996-04-18 |
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